In this renewal application, the second messenger pathways in thrombin receptor activation will be linked to specific locations in the extracellular domain of the receptor, focusing on protein kinase C (PKC) isoforms and myosin light chain kinase (MLCK). The functional effect studied has been increases in vascular endothelial cell permeability in endothelial cell cultures and in rat lung preparations. New information on the sequence and structure of the thrombin receptor will be used to generate antibodies to specific domains and define the "tethered-ligand" mediated receptor activation, as defined by PKC and MLCK activity as well as Ca increase, and inositol polyphosphate generation, and for increasing endothelial cell permeability measured in several ways. PKC isoforms and their relation to endothelial cell permeability will be investigated utilizing gene transfection experiments with PKC alpha or Beta-1 in the sense or antisense orientation to overexpress or inhibit the isoenzymes. MLCK phosphorylation and its linkage to endothelial permeability will be explored with the endothelial cell transfection approaches in similar fashion to that for PKC.